Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry and NMR Analysis Using an External Chamber.

We describe here a method to study and manipulate photorespiration in intact illuminated leaves. When the CO/O mole fraction ratio changes, instant sampling is critical, to quench leaf metabolism and thus trace rapid metabolic modification due to gaseous conditions. To do so, we combine CO labeling and gas exchange, using a large custom leaf chamber to facilitate fast sampling by direct liquid nitrogen spraying. Moreover, the use of a high chamber surface area (about 130 cm) allows one to sample a large amount of leaf material to carry out C-nuclear magnetic resonance (NMR) analysis and complementary analyses, such as isotopic analyses by high-resolution mass spectrometry (by both GC and LC-MS). C-NMR gives access to absolute C amounts at the specific carbon atom position in the labeled molecules and thereby provides an estimate of C-flux of photorespiratory intermediates. Since NMR analysis is not very sensitive and can miss minor metabolites, GC or LC-MS analyses are useful to monitor metabolites at low concentrations. Furthermore, C-NMR and high-resolution LC-MS allow to estimate isotopologue distribution in response to CO labeling while modifying photorespiration activity.